Project Summary CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes) functions as an adaptive immune system in prokaryotes by providing protection against bacterial viruses (phages). CRISPR loci consist of repeat sequences interspersed with short fragments of phage DNA (spacers). The CRISPR-associated (cas) genes code for proteins that regulate 1) the acquisition of new spacers during phage infection (?immunization?) and 2) the use of spacer transcripts to identify and degrade complementary foreign DNA sequences (protospacers) to prevent infection (?immunity?). My proposed research seeks to better understand the functions of the regulatory components of the Streptococcus pyogenes type II-A CRISPR-Cas system (SpyCas9) which is near ubiquitous in gene editing applications. The first aim concerns the protospacer adjacent motifs (PAMs) found in phage DNA. The canonical SpyCas9 PAM sequence is ?NGG?, but non-canonical (non-NGG) PAM sequences are also seen. I collected non-NGG PAMs from spacer acquisition databases and identified those that efficiently mediated immunity. All such PAMs had the sequence ?NAGG?. Thus, I hypothesize that NAGG PAMs can interact with Cas9 to mediate significant immunity. To test this, I will identify the importance of each nucleotide for immunity, as well as the immune potential of NAGGs not seen in the databases. I will also compare AGG and NAGG PAMs via direct competition during phage targeting and via libraries of all possible NAGG and AGG PAMs. Finally, I will biochemically characterize the roles of NAGG PAMs in vitro with DNA binding and cleavage assays. The second aim centers on the enigmatic SpyCas9 protein, Csn2. The other SpyCas9 Cas proteins are well- characterized, but Csn2 is known only to be essential for spacer acquisition in vivo. I investigated Csn2?s impact on the sub-steps of in vivo spacer acquisition from a cleaved protospacer prior to spacer integration into the CRISPR array. The absence of Csn2 in those stages causes a 100-fold decrease in acquisition. Hence, I hypothesize that Csn2 facilitates the selection spacers by interacting with the cleaved protospacer before it is integrated into the CRISPR array. I will test spacer acquisition from WT and ?csn2 strains, evaluating new spacers for length, orientation, acquisition frequency, and their origin (host or foreign DNA). I will use mutagenesis to evaluate the importance of conserved Csn2 residues for spacer acquisition. Lastly, I will determine if Csn2 impacts the selection of acquired spacers by interacting with specific protospacer sequences. The proposed research will uncover essential elements of SpyCas9 functioning by providing a greater understanding of the stringency of PAM recognition and by determining Csn2?s role during spacer acquisition in vivo. These findings have the potential to further fuel the CRISPR revolution in biomedicine, from the tracking of cell ?memories? in pathogens and cancer cells, to the improvement of phage therapies, to the development of exquisitely precise gene editing systems. In doing, my findings will also ultimately transform patients? lives.